The internal organization and dynamics of eukaryotic cells is largely determined by the framework of cytoskeletal elements and molecular motors that transport organelles and macromolecular complexes to specific destinations in the cell. Long range transport is generally mediated by microtubule-based motors, and short range transport and capture by the actin-based motors, myosins. The myosin-V family of molecular motors is evolutionarily highly conserved between animals, plants and fungi, and defects in vertebrate myosin-Vs can cause disease. Many different organelles are transported by myosin-Vs, but how they recognize specific cargo, transport and release it at its destination is poorly understood. To address these questions, the researchers use budding yeast where the essential myosin-V, encoded by Myo2, transports secretory vesicles for growth as its major cargo, but also transports peroxisomes, compartments of the secretory pathway, and the vacuole for segregation during the cell cycle, and microtubule ends for nuclear orientation prior to mitosis. These transport events are all mediated by organelle-specific receptors, many of which are known. Here the researchers explore three fundamental aspects of the Myo2 secretory vesicle delivery cycle. In the first aim, they use quantitative live-cell imagining to quantify the number of motors per secretory vesicle and establish how this is determined, and the kinetics of the delivery cycle and how this is integrated with tethering and fusion of the vesicles at their destination. They also explore mechanisms for recycling and regulation of Myo2. In the second aim, they use genetic and live cell imaging methods to explore the function of two proteins, Mmr1 and Smy1, that bind the tail of Myo2 and seem to play a role in its delivery cycle. In the third aim, they first propose to exploit genetic and biochemical approaches to extend their studies on the receptor by which Myo2 identifies and binds secretory vesicles. They also explore the relationship between binding sites on the Myo2 cargo-binding tail by identifying specific mutations that compromise the interactions with individual receptors, and also exploring how binding to one receptor affects binding to another. Finally, they propose to set up an in vitro assay to reconstitute the binding of Myo2 to secretory vesicles based on the genetic, biochemical and cell biological information collected in the earlier aims. Overall, this study will provide unprecedented insights into the delivery cycle of Myo2, which will be of broad relevance due to the high conservation between fungal and vertebrate myosin-Vs as well as their dysfunction in diseases such as Griscelli's syndrome. PUBLIC HEALTH RELEVANCE: All non-infectious diseases are caused by cellular defects that translate into dysfunction of organ(s). As molecular motors selectively ferry organelles and macromolecular complexes to specific sites in the cell to provide the appropriate cellular organization, this project studies in detail how an evolutionarily conserved motor, myosin-V, picks up, transports and drops off many different specific organelles. This is of crucial importance as the regulated transport of specific cargos, including critical membrane proteins like growth factor receptors, proteins involved in nutrient uptake, and adhesion molecules determine the functions of cells, and defects in these processes contribute to many diseases, including cancer.